1. Field of the Invention
The present invention relates to detection of infectious agents, more specifically, by using oligonucleotide probes, kit containing the same for rapid pathotyping of H5 avian influenza viruses.
2. Description of the Related Art
Avian Influenza virus (AIV) infects many animals such as humans, pigs, horses, marine mammals, and birds. Its natural reservoir is in aquatic birds, and in avian species most influenza virus infections cause mild localized infections of the respiratory and intestinal tract. While the majority of AIV subtypes are classified as low-pathogenicity avian influenza viruses (LPAIV), the H5 and H7 subtypes have the ability to mutate to high pathogenic avian influenza viruses (HPAIV). The HPAIV can have high pathogenic effect in poultry, with sudden outbreaks causing high mortality rates in affected poultry populations. In humans, the HPAIV cause a highly contagious acute respiratory disease that have resulted in epidemic and pandemic disease. Thus, it is of great importance to rapidly detect influenza viruses and promptly discriminate between LPAIV and HPAIV.
Reliable methods for pathotyping of AIV H5 viruses are based on determination of the intravenous pathogenicity index (IVPI) in specific pathogen free (SPF) chickens and on characterization of the hemagglutinin (HA) gene cleavage site (SC) by sequencing. The amino acid motif at the CS of the HA precursor protein of H5 viruses has been found to have a consistent format, PQm-Xn-*GLF (SEQ ID NO: 51) (Leijon et al., Journal of Clinical Microbiology, November 2011, p. 3860-3873) and a large number of Genbank nucleotide analysis (m Glutamine composes the vast majority, although other residues may be seen but few. X: all kinds of residues, in the present invention, it specifically represents arginine and lysine; n: the number of X. *Actual cleavage site at HA0 by host proteases.) The number of basic amino acids (arginine and lysine) at X is an indicator of pathogenicity; in general, LPAIVs have one to three basic amino acids (n=1˜3), and HPAIVs have four or more basic amino acids (n=4 or more).
Currently, there are a variety of techniques that can be used as a pathogenicity test for H5 avian influenza virus in biological samples, including PCR followed by nucleotides sequencing across the CS, real-time PCR, restriction enzyme cleavage pattern of reverse transcription PCR product and the like. However, those methods are time-consuming, labor-intensive, costly and highly complicated to perform. A microarray system, ArayTube, has been reported to pathotype AIV, however, the result interpretation needed the reader and was only limited to parts of H5 viruses, e.g. highly pathogenic H5/N1/Aisa clade 2.2.
Therefore, there remains a need for a lower cost, easier implement, more comprehensive and rapid approach with high sensitivity and specificity.